Topical antibiotics are widely used to reduce surgical site infections in spinal fusion procedures; however, their potential effects on bone healing and fusion-related outcomes remain insufficiently defined, particularly for locally applied glycopeptide antibiotics. Forty-five female Wistar rats underwent unilateral posterolateral lumbar fusion in five groups: control (no antibiotics), low-dose vancomycin (14.3 mg/kg), high-dose vancomycin (71.5 mg/kg), low-dose teicoplanin (15 mg/kg), and high-dose teicoplanin (50 mg/kg). Iliac autograft mixed with antibiotics were evaluated using micro-CT imaging, manual palpation, and histological analysis. Fusion mass volume was significantly reduced in the high-dose vancomycin group compared with the control and teicoplanin-treated groups (p < 0.05). In contrast, micro-CT–based fusion scores did not differ significantly among the groups. Histological analysis demonstrated qualitatively reduced new bone formation and less organized bone architecture in the high-dose vancomycin group, whereas both teicoplanin groups preserved bone morphology comparable to that of the control group. High-dose local vancomycin administration was associated with reduced fusion mass volume and less favorable bone morphology, without significant differences in micro-CT–based fusion scores. In contrast, both low- and high-dose teicoplanin preserved bone integrity comparable to controls, suggesting that the type and dosage of locally applied antibiotics may influence fusion mass characteristics and should be considered alongside antimicrobial efficacy in spinal fusion procedures.